Sexual selection is among the most powerful of all evolutionary forces. It occurs when individuals within one sex secure mates and produce offspring at the expense of other individuals within the same sex. Darwin was first to recognize the power of sexual selection to change male and female phenotypes, and, in noting that sexual selection is nonubiquitous, Darwin was also first to recognize the importance of mating system--the "special circumstances" in which reproduction occurs within species. Analyses of mating systems since Darwin have emphasized either the genetic relationships between male and female mating elements, usually among plants, or the numbers of mates males and females may obtain, usually among animals. Combining these schemes yields a quantitative methodology that emphasizes measurement of the sex difference in the variance in relative fitness, as well as phenotypic and genetic correlations underlying reproductive traits that may arise among breeding pairs. Such information predicts the degree and direction of sexual dimorphism within species, it allows the classification of mating systems using existing genetic and life history data, and with information on the spatial and temporal distributions of fertilizations, it may also predict floral morphology in plants. Because this empirical framework identifies selective forces and genetic architectures responsible for observed male-female differences, it compliments discoveries of nucleotide sequence variation and the expression of quantitative traits. Moreover, because this methodology emphasizes the process of evolutionary change, it is easier to test and interpret than frameworks emphasizing parental investment in offspring and its presumed evolutionary outcomes.

BACKGROUND

Theobroma cacao, the chocolate tree, is an important economic crop in East Africa, South East Asia, and South and Central America. Propagation of elite varieties has been achieved through somatic embryogenesis (SE) but low efficiencies and genotype dependence still presents a significant limitation for its propagation at commercial scales. Manipulation of transcription factors has been used to enhance the formation of SEs in several other plant species. This work describes the use of the transcription factor Baby Boom (BBM) to promote the transition of somatic cacao cells from the vegetative to embryonic state.

RESULTS

An ortholog of the Arabidopsis thaliana BBM gene (AtBBM) was characterized in T. cacao (TcBBM). TcBBM expression was observed throughout embryo development and was expressed at higher levels during SE as compared to zygotic embryogenesis (ZE). TcBBM overexpression in A. thaliana and T. cacao led to phenotypes associated with SE that did not require exogenous hormones. While transient ectopic expression of TcBBM provided only moderate enhancements in embryogenic potential, constitutive overexpression dramatically increased SE proliferation but also appeared to inhibit subsequent development.

CONCLUSIONS

Our work provides validation that TcBBM is an ortholog to AtBBM and has a specific role in both somatic and zygotic embryogenesis. Furthermore, our studies revealed that TcBBM transcript levels could serve as a biomarker for embryogenesis in cacao tissue. Results from transient expression of TcBBM provide confirmation that transcription factors can be used to enhance SE without compromising plant development and avoiding GMO plant production. This strategy could compliment a hormone-based method of reprogramming somatic cells and lead to more precise manipulation of SE at the regulatory level of transcription factors. The technology would benefit the propagation of elite varieties with low regeneration potential as well as the production of transgenic plants, which similarly requires somatic cell reprogramming.

OBJECTIVE

Anorexia nervosa (AN) patients tend to place a positive value on their symptoms. Many clinicians believe that this plays a central role in maintaining the disorder. However, empirical research on how patients attribute meaning to their symptoms is lacking. This study aims at systematically exploring the meaning that the patients with AN attribute to their anorectic behavior.

METHODS

A qualitative, descriptive, phenomenological design was used. Eighteen women aged 20-34 with AN (DSM-IV) were interviewed with an informant-centered interview. The interviews were tape-recorded, verbatim transcribed, coded, and analyzed phenomenologically, using a QSR-N*Vivo software program.

RESULTS

The psychological meanings that the informants attributed to their anorectic behavior could be summarized in eight constructs: "Security" (feeling of stability and security), "Avoidance" (avoiding negative emotions), "Mental strength" (inner sense of mastery), "Self-confidence" (feeling acknowledged and worthy of compliments); "Identity" (achieving new identity), "Care" (eliciting care from others), "Communication" (communicating difficulties), and "Death" (wishing to starve oneself to death).

CONCLUSIONS

The eight constructs may have central functions in the maintenance of AN and should be regarded when patients' motivation and goals for treatment are assessed. Further study of the possible functions of the constructs in maintaining AN is warranted.

OBJECTIVE

In neonatal lambs, the quantitative evidence suggests that a significant volume of cranial CSF drainage is associated with transport along olfactory nerves with absorption primarily into extracranial lymphatics in the paranasal region. Arachnoid granulations appear to be poorly developed at this level of development and their function is unknown. In this report, we tested whether a CSF protein tracer ((131)I-human serum albumin) could transport directly into the superior sagittal sinus of newborn lambs.

RESULTS

The concentration of the tracer administered into the CSF compartment was measured in the confluence of the intracranial venous sinuses (torcula) and in the peripheral blood (inferior vena cava). Enrichment of the CSF tracer in the cranial venous system was most evident when the CSF-venous sinus pressure gradients approached 20-30 cm H(2)O.

CONCLUSIONS

The data suggests that neonatal CSF can be absorbed directly into the cranial venous system. However, contrary to the classical view, this route may represent an auxiliary system that is recruited to compliment lymphatic transport when intracranial pressures are very high.

Non-invasive in-vivo molecular genetic imaging developed over the past decade and predominantly utilises radiotracer (PET, gamma camera, autoradiography), magnetic resonance and optical imaging technology. Molecular genetic imaging has its roots in both molecular biology and cell biology. The convergence of these disciplines and imaging modalities has provided the opportunity to address new research questions, including oncogenesis, tumour maintenance and progression, as well as responses to molecular-targeted therapy. Three different imaging strategies are described: (1) "bio-marker" or "surrogate" imaging; (2) "direct" imaging of specific molecules and pathway activity; (3) "indirect" reporter gene imaging. Examples of each imaging strategy are presented and discussed. Several applications of PET- and optical-based reporter imaging are demonstrated, including signal transduction pathway monitoring, oncogenesis in genetic mouse models, endogenous molecular genetic/biological processes and the response to therapy in animal models of human disease. Molecular imaging studies will compliment established ex-vivo molecular-biological assays that require tissue sampling by providing a spatial and a temporal dimension to our understanding of disease development and progression, as well as response to treatment. Although molecular imaging studies are currently being performed primarily in experimental animals, we optimistically expect they will be translated to human subjects with cancer and other diseases in the near future.

Common genetic disorders are believed to arise from the combined effects of multiple inherited genetic variants acting in concert with environmental factors, such that any given DNA sequence variant may have only a marginal effect on disease outcome. As a consequence, the correlation between disease status and any given DNA marker allele in a genomewide linkage study tends to be relatively weak and the implicated regions typically encompass hundreds of positional candidate genes. Therefore, new strategies are needed to parse relatively large sets of 'positional' candidate genes in search of actual disease-related gene variants. Here we use biological databases to identify 383 positional candidate genes predicted by genomewide genetic linkage analysis of a large set of families, each with two or more members diagnosed with autism, or autism spectrum disorder (ASD). Next, we seek to identify a subset of biologically meaningful, high priority candidates. The strategy is to select autism candidate genes based on prior genetic evidence from the allelic association literature to query the known transcripts within the 1-LOD (logarithm of the odds) support interval for each region. We use recently developed bioinformatic programs that automatically search the biological literature to predict pathways of interacting genes (PATHWAYASSIST and GENEWAYS). To identify gene regulatory networks, we search for coexpression between candidate genes and positional candidates. The studies are intended both to inform studies of autism, and to illustrate and explore the increasing potential of bioinformatic approaches as a compliment to linkage analysis.

A number of empirical studies have documented the relationship between quantifiable and objective acoustical measures of voice and speech, and clinical subjective ratings of severity of Major Depression. To further explore this relationship, speech samples were extracted from videotape recordings of structured interviews made during the administration of the 17-item Hamilton Depression Rating Scale (HDRS; ). Pilot data were obtained from seven subjects (five males, two females) from videotapes that have been used to train expert raters on the administration and scoring of the HDRS. Several speech samples were isolated for each subject and processed to obtain the acoustic measurements. Acoustic measures were selected on the basis that they were correlated with HDRS ratings of symptom severity as seen under ideal voice recording conditions in previous studies. Our findings corroborate earlier reports that speaking rate is well correlated (negatively) with HDRS scores, with a strong correlation and nearly significant trend seen for the measure of pitch variability. A moderate pairwise correlation between percent pause time and HDRS score was also revealed, although this relationship was not statistically significant. The results from this cross-sectional study further demonstrate the ability of voice and speech signal analyses to objectively track severity of depression. In the present case, it is suggested that this relationship is robust enough to be found despite the less than ideal recording conditions and equipment used during the original videotape recording. Voice acoustical analyses may provide a powerful compliment to the standard clinical interview for depression. Use of such measures increases the range of techniques that are available to explore the neurobiological substrates of Major Depression, its treatment, and the dynamic interplay of the systems that govern the motor, cognitive, and emotional aspects of speech production.

OBJECTIVE

Health information technology has been shown to influence the communication patterns of healthcare providers. The goal of this study was to learn more about how healthcare providers communicate and exchange patient clinical information during patient handoffs (transfers) between units in an acute care setting.

METHODS

Convenience sampling was used to select five patient handoffs. Questionnaires were distributed to providers identified through observation and snowball sampling. Social network analysis methodology was used to develop sociograms of the emergent communication patterns and identify the role of individual providers in the handoff process based on the number of contacts with other providers and incoming and outgoing communication activity. Individual handoff network size ranged from 11 to 20 providers. Participants were asked to describe the method of communication they used to access or share clinical information with other providers, their preferred method of communication; their satisfaction with the available options; and their suggestions for how the process could be improved.

RESULTS

The network patterns that emerged uncovered the overlapping use of synchronous and asynchronous communication methods (verbally via phone or in person; or written via paper charts and/or an electronic records). No particular professional group dominated or coordinated information flow; instead each handoff network exhibited unique communication patterns and information coordination by two or more influential providers from nursing, medicine, or pharmacy. Most (84%) participants preferred verbal communication. Overall satisfaction with the current communication process varied by unit: 82% of emergency department providers and 54% of the providers working in the admitting units stated they were satisfied or very satisfied. Recommendations for improvement included converting all units to the electronic health record, electronic handoff communication modules and asynchronous multi-professional communication logs.

CONCLUSIONS

The results of this exploratory study provide a foundation for future research examining how network structure and communication principles can be used to design health information technology that compliments the non-linear information gathering and dissemination behaviors of providers from multiple professions.

Microarray analysis of the genome of Lactobacillus acidophilus identified a number of operons that were differentially expressed in response to carbohydrate source or constitutively expressed regardless of carbohydrate source. These included operons implicated in the transport and catabolism of fructooligosaccharides (FOS), lactose (lac), trehalose (tre) and genes directing glycolysis. Analysis of these operons identified a number of putative promoter and repressor elements, which were used to construct a series of expression vectors for use in lactobacilli, based on the broad host range pWV01 replicon. A β-glucuronidase (GusA3) reporter gene was cloned into each vector to characterize expression from each promoter. GUS reporter assays showed FOS, lac and tre based vectors to be highly inducible by their specific carbohydrate and repressed by glucose. Additionally, a construct based on the phosphoglycerate mutase (pgm) promoter was constitutively highly expressed. To demonstrate the potential utility of these vectors, we constructed a plasmid for the overexpression of the oxalate degradation pathway (Frc and Oxc) of L. acidophilus NCFM. This construct was able to improve oxalate degradation by L. gasseri ATCC 33323 and compliment a L. acidophilus oxalate-deficient mutant. Development of these expression vectors could support several novel applications, including the expression of enzymes, proteins, vaccines and biotherapeutics by intestinal lactobacilli.

BACKGROUND

New mathematical models of complex biological structures and computer simulation software allow modelers to simulate and analyze biochemical systems in silico and form mathematical predictions. Due to this potential predictive ability, the use of these models and software has the possibility to compliment laboratory investigations and help refine, or even develop, new hypotheses. However, the existing mathematical modeling techniques and simulation tools are often difficult to use by laboratory biologists without training in high-level mathematics, limiting their use to trained modelers.

RESULTS

We have developed a Boolean network-based simulation and analysis software tool, ChemChains, which combines the advantages of the parameter-free nature of logical models while providing the ability for users to interact with their models in a continuous manner, similar to the way laboratory biologists interact with laboratory data. ChemChains allows users to simulate models in an automatic fashion under tens of thousands of different external environments, as well as perform various mutational studies.

CONCLUSIONS

ChemChains combines the advantages of logical and continuous modeling and provides a way for laboratory biologists to perform in silico experiments on mathematical models easily, a necessary component of laboratory research in the systems biology era.

BACKGROUND

Culture of M. bovis from diagnostic specimens is the gold standard for bovine tuberculosis diagnostics in the USA. Detection of M. bovis by PCR in tissue homogenates may provide a simple rapid method to complement bacterial culture. A significant impediment to PCR based assays on tissue homogenates is specificity since mycobacteria other than M. bovis may be associated with the tissues.

RESULTS

Previously published IS6110 based PCR diagnostic assays, along with one developed in house, were tested against environmental mycobacteria commonly isolated from diagnostic tissues submitted to the National Veterinary Services Laboratory. A real-time PCR assay was developed (IS6110_T) that had increased specificity over other IS6110 based assays. Of the 13 non-tuberculous mycobacteria tested with IS6110_T only M. wolinskyi was positive. Thirty M. bovis infected tissue homogenates and 18 control tissues were used to evaluate the potential for the assay as a diagnostic test. In this small sample, IS6110_T detected 20/30 samples from M. bovis infected animals and 0/18 control tissues.

CONCLUSIONS

The IS6110_T assay provides a PCR based assay system that is compatible with current diagnostic protocols for the detection of M. bovis in the USA and compliments current testing strategies.

The aspartic protease pepsin is less specific than other endoproteinases. Because aspartic proteases like pepsin are active at low pH, they are utilized in hydrogen deuterium exchange mass spectrometry (HDX MS) experiments for digestion under hydrogen exchange quench conditions. We investigated the reproducibility, both qualitatively and quantitatively, of online and offline pepsin digestion to understand the compliment of reproducible pepsin fragments that can be expected during a typical pepsin digestion. The collection of reproducible peptides was identified from >30 replicate digestions of the same protein and it was found that the number of reproducible peptides produced during pepsin digestion becomes constant above 5-6 replicate digestions. We also investigated a new aspartic protease from the stomach of the rice field eel (Monopterus albus Zuiew) and compared digestion efficiency and specificity to porcine pepsin and aspergillopepsin. Unique cleavage specificity was found for rice field eel pepsin at arginine, asparagine, and glycine. Different peptides produced by the various proteases can enhance protein sequence coverage and improve the spatial resolution of HDX MS data. This article is part of a Special Issue entitled: Mass spectrometry in structural biology.

Locus ceruleus (LC) noradrenergic neurons are critical in generating alertness. In addition to inducing cortical arousal, the LC also orchestrates changes in accompanying autonomic system function that compliments increased attention, such as during stress, excitation, and/or exposure to averse or novel stimuli. Although the association between arousal and increased heart rate is well accepted, the neurobiological link between the LC and parasympathetic neurons that control heart rate has not been identified. In this study, we test directly whether activation of noradrenergic neurons in the LC influences brainstem parasympathetic cardiac vagal neurons (CVNs). CVNs were identified in transgenic mice that express channel-rhodopsin-2 (ChR2) in LC tyrosine hydroxylase neurons. Photoactivation evoked a rapid depolarization, increased firing, and excitatory inward currents in ChR2-expressing neurons in the LC. Photostimulation of LC neurons did not alter excitatory currents, but increased inhibitory neurotransmission to CVNs. Optogenetic activation of LC neurons increased the frequency of isolated glycinergic IPSCs by 27 ± 8% (p = 0.003, n = 26) and augmented GABAergic IPSCs in CVNs by 21 ± 5% (p = 0.001, n = 26). Inhibiting α1, but not α2, receptors blocked the evoked responses. Inhibiting β1 receptors prevented the increase in glycinergic, but not GABAergic, IPSCs in CVNs. This study demonstrates LC noradrenergic neurons inhibit the brainstem CVNs that generate parasympathetic activity to the heart. This inhibition of CVNs would increase heart rate and risks associated with tachycardia. The receptors activated within this pathway, α1 and/or β1 receptors, are targets for clinically prescribed antagonists that promote slower, cardioprotective heart rates during heightened vigilant states.

OBJECTIVE

We report diverse phenotypic consequences of the delQKP-1507-1509 cardiac sodium channel mutation in three generations of a Chinese family.

RESULTS

Clinical and electrocardiographic (ECG), echocardiographic examination was followed by direct sequencing of SCN5A, KCNQ1, HERG, and LAMIN A/C to screen genomic DNA from blood samples. Of two mutation carriers, the proband was born with conduction disorders including second-degree atrioventricular (AV) block with prolonged QTc interval, additionally showing left anterior fascicular block (LAFB), incomplete right bundle-branch block (IRBBB), and intermittent third-degree AV block at 2 years, and clinical presentations of multiple syncope despite normal electroencephalograms at 8 years. Continuous ECG monitoring following presentation at 13 years revealed prolonged QTc and biphasic T-waves, multiple episodes of ventricular tachycardia, ventricular fibrillation, and torsades de pointes. Transthoracal echocardiography then revealed left ventricular dilatation and reduced systolic function. Another mutation carrier showed features of long QT syndrome type 3 (LQT3), LAFB, and dilated cardiomyopathy (DCM). Two additional subjects died suddenly at 13 and 33 years.

CONCLUSIONS

This data compliments and expands the spectrum of phenotypes resulting from this known gain-of-function mutation, including not only LQT3, cardiac conduction defects, and sudden death but also DCM, hitherto associated with loss-of-function mutations, for the first time.

OBJECTIVE

The purpose of this study was to develop a measure of psychosocial barriers to adherence in adolescents with type 1 diabetes (T1D) and examine relationships to patient characteristics, adherence, and hemoglobin A1C (A1C).

METHODS

Barriers to diabetes adherence (BDA) items were generated by researchers, clinicians, and patients. Adolescents aged 12-17 with T1D completed the BDA and an adherence measure. Hemoglobin A1C was obtained through medical chart review.

RESULTS

Factor analysis from 123 adolescents resulted in a 21-item, five-component solution that accounted for 64.5% of the variance. The components were stress and burnout, time pressure and planning, social support, parental autonomy support, and stigma. The BDA total and subscales were internally consistent. The BDA total and some components were associated with adherence and A1C. The BDA was the only predictor of A1C compared to demographic, clinical, and adherence variables (F 6.17, p<.05). Subjects with higher A1C (>8.5) showed a higher level of barriers (F 15.20, p<.001) and a differential profile of barriers (F 5.75, p<.05).

CONCLUSIONS

The BDA may be useful in research and clinical settings as a compliment to adherence measures and to tailor educational programs. Additional research is necessary to establish test-retest reliability and discriminant validity.

BACKGROUND

Developed countries are experiencing a dramatic increase in the proportion of elderly persons, as well as a progressive aging of the elderly population itself. Knowledge regarding the amount of formal and informal care and its interaction at population-based level is limited.

OBJECTIVE

To describe the amount of formal and informal care for non-demented and demented persons living at home in a population-based sample.

METHODS

The population consisted of all inhabitants, 75 + years, living in a rural community (n = 740). They were clinically examined by physicians and interviewed by nurses. Dementia severity was measured according to Washington University Clinical Dementia Rating Scale (CDR). Informal and formal care was examined with the RUD (Resource Utilization in Dementia) instrument.

RESULTS

The amount of informal care was much greater than formal care and also greater among demented than non-demented. There was a relationship between the severity of the cognitive decline and the amount of informal care while this pattern was weaker regarding formal care. Tobit regression analyses showed a clear association between the number of hours of informal and formal care and cognitive decline although this pattern was much stronger for informal than formal care.

CONCLUSIONS

Informal care substitutes rather than compliments formal care and highlights the importance of future studies in order to truly estimate the amount of informal and formal care and the interaction between them. This knowledge will be of importance when planning the use of limited resources, and when supporting informal carers in their effort to care for their intimates.

BACKGROUND

Anti-Wolbachia treatment with doxycycline is effective in sterilising and killing adult Onchocerca volvulus nematodes, proving superior to ivermectin and of great potential as an alternative approach for the treatment and control of onchocerciasis, particularly in areas of Loa loa co-endemicity. Nevertheless, the length of the required treatment poses potential logistical problems and risk of poor compliance, raising a barrier to the use of doxycycline in Mass Drug Administration (MDA) strategies. In 2007 and 2008 a feasibility trial of community-directed treatment with doxycycline was carried out in two health districts in Cameroon, co-endemic for O. volvulus and L. loa. With 17,519 eligible subjects, the therapeutic coverage was 73.8% with 97.5% compliance, encouraging the feasibility of using doxycycline community-directed delivery in restricted populations of this size. The current study evaluated the effectiveness of this community-directed delivery of doxycycline four years after delivery.

RESULTS

Infection with O. volvulus was evaluated by skin biopsy and nodule palpation. Of the 507 subjects recruited, 375 had completed the treatment with doxycycline followed by one or two rounds of annual ivermectin MDA and 132 received one or two rounds of annual ivermectin MDA alone. Statistically significant lower microfilarial prevalence (17.0% [doxycycline plus ivermectin group], 27.0% [ivermectin only group], p = 0.014) and load (p = 0.012) were found in people that had received doxycycline followed by ivermectin compared to those who received ivermectin only.

CONCLUSIONS

This study demonstrates the long-term effectiveness of doxycycline treatment delivered with a community-directed strategy even when evaluated four years after delivery in an area of ongoing transmission. This finding shows that a multi-week course of treatment is not a barrier to community-delivery of MDA in restricted populations of this size and supports its implementation to compliment existing control strategies for onchocerciasis, where needed.

HMG-CoA reductase inhibitors (statins) reduce cardiovascular disease morbidity and mortality with a high level of safety. Nonetheless, there are substantial numbers of people who either do not tolerate statins or whose low-density lipoprotein (LDL) levels are not lowered adequately. For these reasons, there is a need to develop other cholesterol-lowering drugs. A target for these new agents is provided by the enzymes distal to HMG-CoA reductase in the cholesterol biosynthesis pathway. Two classes of drugs have been developed: (i) squalene synthase inhibitors, which act at the first committed step in cholesterol biosynthesis, distal to the mevalonate-farnesyl diphosphate pathway; and (ii) oxidosqualene cyclase inhibitors, which act distal to the squalene intermediate. Of these, squalene synthase inhibitors have received more attention and are the subject of this review. Squalene synthase inhibitors decrease circulating LDL-cholesterol by the induction of hepatic LDL receptors in a similar manner to statins. They have fewer secondary effects mediated by a decrease in non-cholesterol products of mevalonate metabolism distal to HMG-CoA reductase, but have the potential to increase intermediates proximal to squalene. Squalene synthase inhibitors are just now entering clinical trials and data on how effectively they lower LDL-cholesterol and how they compliment the actions of statins and other agents is awaited with considerable interest.

Hematogeneous spread of primary neoplasm can result in central nervous system (CNS) disease burden in various anatomically distinct regions; calvarial, pachymeningeal, leptomeningeal, and intraparenchymal. The choice of imaging modality is dependent on the individual clinical situation, but, largely depends on the patients overall clinical status and the information needed to make treatment decisions. Contrast-enhanced magnetic resonance (MR) imaging is the preferred imaging modality of choice; however, computed tomography (CT) is often utilized as the first-pass screening modality for CNS disease. Despite the superior soft tissue resolution, multiplanar capability, and noninvasive nature of MR imaging, T(1)- and T(2)-weighted sequences are limited to delineating morphologic anatomical deraignment of tissues by tumor. Several physiology based MR imaging sequences have been developed which compliment anatomic MR imaging. Proton magnetic resonance spectroscopic and dynamic susceptibility contrast-enhanced perfusion-weighted imaging are two physiologic sequences which add additional diagnostic information allowing for improved tumor characterization. Common pitfalls in evaluating for metastatic disease burden include the misidentification of non-neoplastic hematomas, remote microvascular ischemia, and acute onset of ischemic stroke. In the pediatric population, CNS metastases are rare; however, the onset of acute neurological symptoms in a child with known primary tumor should prompt imaging of the neuroaxis.

Contrary to what occurs with negative pictures, negative words are, in general, not capable of interfering with performance in ongoing cognitive tasks in normal subjects. A probable explanation is the limited arousing power of linguistic material. Especially intense words (insults and compliments), neutral personal adjectives, and pseudowords were presented to 28 participants while they executed a lexical decision task. Insults were associated with the poorest performance in the task and compliments with the best. Amplitude of the late positive component of the event-related potentials, originating at parietal areas, was maximal in response to compliments and insults, but latencies were delayed in response to the latter. Results suggest that intense emotional words modulate ongoing cognitive processes through both bottom-up (attentional capture by insults) and top-down (facilitation of cognitive processing by arousing words) mechanisms.

A clear understanding of the mechanism of function of immune stimulatory adjuvants, which commonly accompany vaccines, is beginning to emerge. Recent investigations have demonstrated that Toll-like receptors (TLRs) are the critical link between the innate and the adaptive immunity. This link, which is normally activated as a result of collaboration between adjuvants and TLRs in triggering adaptive immunity, has been a subject of several recent investigations. With the advent of well-defined synthetic small molecules, which are designed to either mimic the adjuvants or, as in many cases, to structurally represent pathogen associated molecular patterns, it is now possible to design reproducible experiments and to draw credible conclusions. An adjuvant alerts the host immune system through a mechanism similar to that of an infection by a pathogen, which involves interaction with a TLR followed by a lsqou;danger signal' to the immune system. Secretion of cytokines and regulation of the expression of co-stimulatory molecules induced by innate response shape the magnitude and quality of adaptive response. Synthetic vaccines containing specific epitopes to which immune responses are desired, are expected to be far superior in target specificity while the benefits may be long-lasting. The immune responses by therapeutic vaccines are generally adaptive in nature and such responses often require the participation of the components of innate immunity, most importantly the TLRs and their pathogen-associated binding compliments. Structurally well-defined synthetic molecules derived from lipid A, muramyl di-peptide (MDP), and CpG motifs from bacterial DNA offer a wide range of immune stimulants for the development of fully synthetic vaccines. Lipo-peptide and self-adjuvanted antigens, in combination with additional immune stimulatory adjuvants in liposome delivery system, may be important in vaccine design. Combinations of synthetic mimics of microbial products are known to display synergistic effects in stimulating the immune system. Either alone or in combination with chemotherapy, innate immune therapy using TLR ligands to stimulate the immune system may offer an alternate therapeutic approach against rapidly mutating viral infections-(HIV/AIDS), and cancers.

Classical techniques for estimating postsynaptic potentials in motoneurones include spike-triggered averages of rectified surface and multiunit electromyographic recordings (SEMG and MU-EMG), as well as the compilation of peristimulus time histograms (PSTH) based on the discharge of single motor units (SMU). These techniques rely on the probability of spike occurrence in relation to the stimulus and can be contaminated by count- and synchronization-related errors, arising from post-spike refractoriness and the discharge statistics of motoneurones. On the other hand, since these probability-based techniques are easy to use and require only inexpensive equipment, it is very likely that they will continue to be used in clinical and laboratory settings for the foreseeable future. One aim of the present study was to develop a modification of these probability-based analyses in order to provide a better estimate of the initial phase of postsynaptic potentials. An additional aim was to combine probability-based analyses with frequency-based analyses to provide a more reliable estimate of later phases of postsynaptic potentials. To achieve these aims, we have injected simple as well as complex current transients into regularly discharging hypoglossal motoneurones recorded in vitro from rat brainstem slices. We examined the discharge output of these cells using both probability- and frequency-based analyses to identify which of the two represented the profile of the postsynaptic potential more closely. This protocol was designed to obtain PSTHs of the responses of single motor units to repeated application of the same afferent input. We have also simulated multiunit responses to afferent input by replacing the times of spike occurrence in individual trials with a representation of either an intramuscular or surface-recording single motor unit waveform and summing many of these trials to obtain either a simulated SEMG or MU-EMG. We found that in a regularly discharging motoneurone, the rising phase of an EPSP moves the occurrence of spikes forward and hence induces a substantial peak in all probability-based records. This peak is followed immediately by a period of reduced activity ('silent period') due to the phase advancement of spikes that were to occur at this period. Similarly, the falling phase of an IPSP delays spikes so that they occur during the rising phase of the IPSP. During the delay, the probability-based analyses display gaps and during the occurrence of the delayed spikes they generate peaks. We found that all the probability-based analyses (SEMG, MU-EMG and PSTH) can be made useful for illustrating the underlying initial PSP by a special use of the cumulative sum (CUSUM) calculation. We have illustrated that, in most cases, the CUSUM of probability-based analyses can overcome the delay- or advance-related (i.e. the count-related) errors of the classical methods associated with the first PSP only. The probability-based records also induce secondary and tertiary peaks and troughs due to synchronization of the spikes in relation to the stimulus (i.e. the synchronization-related errors) by the first PSP to occur at fixed times from the stimulus. Special CUSUM analyses cannot overcome these synchronization-related errors. Frequency-based analysis (PSFreq) of individual and summed trials gave comparable and often better indications of the underlying PSPs than the probability-based analyses. When used in combination, these analyses compliment each other so that a more accurate estimation of the underlying PSP is possible. Since the correct identification of the connections in the central nervous system is of utmost importance in order to understand the operation of the system, we suggest that as well as the using the special CUSUM approach on probability-based records, researchers should seriously consider the use of frequency-based analyses in their indirect estimation of stimulus-induced compound synaptic potentials in human motoneurones.

The closing conformational transition of wild-type polymerase beta bound to DNA template/primer before the chemical step (nucleotidyl transfer reaction) is simulated using the stochastic difference equation (in length version, "SDEL") algorithm that approximates long-time dynamics. The order of the events and the intermediate states during pol beta's closing pathway are identified and compared to a separate study of pol beta using transition path sampling (TPS) (Radhakrishnan, R.; Schlick, T. Proc. Natl. Acad. Sci. USA 2004, 101, 5970-5975). Results highlight the cooperative and subtle conformational changes in the pol beta active site upon binding the correct substrate that may help explain DNA replication and repair fidelity. These changes involve key residues that differentiate the open from the closed conformation (Asp192, Arg258, Phe272), as well as residues contacting the DNA template/primer strand near the active site (Tyr271, Arg283, Thr292, Tyr296) and residues contacting the beta and gamma phosphates of the incoming nucleotide (Ser180, Arg183, Gly189). This study compliments experimental observations by providing detailed atomistic views of the intermediates along the polymerase closing pathway and by suggesting additional key residues that regulate events prior to or during the chemical reaction. We also show general agreement between two sampling methods (the stochastic difference equation and transition path sampling) and identify methodological challenges involved in the former method relevant to large-scale biomolecular applications. Specifically, SDEL is very quick relative to TPS for obtaining an approximate path of medium resolution and providing qualitative information on the sequence of events; however, associated free energies are likely very costly to obtain because this will require both successful further refinement of the path segments close to the bottlenecks and large computational time.

BACKGROUND

The advent of panneuroendocrine markers has helped to better depict the heterogeneity of gastrointestinal carcinoids. Consequently, it has been proposed that these tumors constitute a histologic spectrum that includes well-, moderately, and poorly differentiated carcinoids. However, the reproducibility of this grading system and its prognostic importance have sometimes been called into question.

OBJECTIVE

To investigate the potential utility of cell proliferation and oncoprotein markers in augmenting the histologic classification.

METHODS

Retrospective study; tertiary care teaching hospital.

METHODS

Fifty-eight patients with 41 well-differentiated, 12 moderately differentiated and 5 poorly differentiated carcinoids from various topographic sites of the gastrointestinal tract were selected and immunostained for panneuroendocrine markers, MIB-1, p53, and bcl-2.

METHODS

Degree of association between histologic grading, MIB-1, p53, and bcl-2 immunoreactivity and carcinoid metastatic behavior.

RESULTS

The group comprised 30 males and 28 females whose mean age was 52.7 years (range, 14-81). Mean follow-up time was 85.8 months. All 58 patients tested positive for chromogranin A and/or synaptophysin. The group was divided into nonmetastatic (42/58, or 72.4%) and metastatic (16/58, or 27.6%) cases. Histologic grading tended to be associated with metastatic spread, but this occurrence of metastases did not attain statistical significance (P =.08). Positivity for MIB-1 (P =.004) and p53 (P =.04) was significantly associated with metastatic behavior, whereas bcl-2 was not (P = 0. 63).

CONCLUSIONS

Although an organoid pattern and neuroendocrine immunophenotype help to define the spectrum of gastrotestinal carcinoids, this study suggests that the histologic grading of these tumors has some limitations with respect to predicting metastatic behavior. However, MIB-1 and p53 can compliment histologic grading as prognostic indicators in this regard while bcl-2 appears to be less useful.